Photosystem II single crystals studied by transient EPR: the light-induced triplet state

Transient electron paramagnetic resonance (TR EPR) at 9.8 GHz has been used to study the light-induced triplet state in single crystals of Photosystem II (PS II). The crystals were grown from a solution of PS II core complexes from the thermophilic cyanobacterium Synechococcus elongatus. The core complexes contain at least 17 subunits, including the water-oxidizing complex, and 32 chlorophyll a molecules per PS II complex. The PS II complexes are active in light-induced electron transfer and water oxidation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with four dimers of PS II complexes per unit cell. Laser excitation was used to generate the recombination triplet state in PS II which was then studied by EPR at low temperatures (10 K). The crystal spectra show the same magnitude of the zero-field splitting (ZFS) values D, E as spectra obtained earlier for the triplet state of PS II in frozen solution. The orientation of the ZFS tensor D of the triplet state with respect to the crystallographic axes has been deduced from the analysis of angular-dependent EPR spectra. Knowledge of the orientation of the D tensor component perpendicular to the plane of the chlorophyll (D(Z)) allows an assignment on which chlorophyll of the reaction centre the triplet state is localized at low temperatures. Furthermore, the orientation of the D(X) and D(Y) components of the D tensor yielded the in-plane orientation of the respective chlorophyll in the reaction centre providing first experimental evidence for the orientation of this molecule in the PS II.     

Quantitative analysis of three-dimensional-resolved fiber architecture in heterogeneous skeletal muscle tissue using nmr and optical imaging methods

The determination of principal fiber directions in structurally heterogeneous biological tissue substantially contributes to an understanding of its mechanical function in vivo. In this study we have depicted structural heterogeneity through the model of the mammalian tongue, a tissue comprised of a network of highly interwoven fibers responsible for producing numerous variations of shape and position. In order to characterize the three-dimensional-resolved microscopic myoarchitecture of the intrinsic musculature of the tongue, we viewed its fiber orientation at microscopic and macroscopic length scales using NMR (diffusion tensor MRI) and optical (two-photon microscopy) imaging methods. Diffusion tensor imaging (DTI) of the excised core region of the porcine tongue resulted in an array of 3D diffusion tensors, in which the leading eigenvector corresponded to the principal fiber orientation at each location in the tissue. Excised axially oriented lingual core tissues (fresh or paraffin-embedded) were also imaged with a mode-locked Ti-Sapphire laser, (76 MHz repetition rate, 150 femtosecond pulse width), allowing for the visualization of individual myofibers at in situ orientation. Fiber orientation was assessed by computing the 3D autocorrelation of discrete image volumes, and deriving the minimal eigenvector of the center voxel Hessian matrix. DTI of the fibers, comprising the intrinsic core of the tongue, demonstrated directional heterogeneity, with two distinct populations of fibers oriented orthogonal to each other and in-plane to the axial perspective. Microscopic analysis defined this structural heterogeneity as discrete regions of in-plane parallel fibers, with an angular separation of ~80 degrees, thereby recapitulating the macroscopic angular relationship. This analysis, conceived at two different length scales, demonstrates that the lingual core is a spatially complex tissue, composed of repeating orthogonally oriented and in-plane fiber patches, which are capable of jointly producing hydrostatic elongation and displacement.     

Orientational order and dynamics of hydration water in a single crystal of bovine pancreatic trypsin inhibitor.

The orientational order and dynamics of the water molecules in form II crystals of bovine pancreatic trypsin inhibitor (BPTI) are studied by (2)H NMR in the temperature range 6-50 degrees C. From the orientation dependence of the single crystal quadrupole splitting and linewidth, the principal components of the motionally averaged quadrupole interaction tensor and the irreducible linewidth components for the orthorhombic crystal are determined. With the aid of water orientations derived from neutron and x-ray diffraction, it is shown that the NMR data can be accounted for by a small number of highly ordered crystal waters, some of which have residence times in the microsecond range. Most of these specific hydration sites must be located at intermolecular contacts. The surface hydration layer that is also present in dilute solution is likely to be only weakly ordered and would then not contribute significantly to the splitting and linewidth from the protein crystal. To probe water dynamics on shorter time scales, the (2)H longitudinal relaxation dispersion is measured for a polycrystalline BPTI sample. The observed dispersion is dominated by rapidly exchanging deuterons in protein side chains, undergoing restricted rotational motions on a time scale of 10 ns.     

Effect of nucleotides and their analogues on essential light chains in myosin head

The domain movement in myosin head plays a decisive role in the energy transduction process of the muscle contraction. During hydrolysis of ATP, the specific formation of strong binding of myosin head for actin causes conformational changes. As a consequence, the light chain-binding motif generates the powerstroke. In our work maleimide spin labels were covalently attached to Cys-177 residue of ELC in subfragment-1 (S1). Our goal was to study the orientation dependence and the motion of S1, which were incorporated into glycerinated skeletal muscle fibres. The electron paramagnetic resonance spectroscopy (EPR) spectra of the probes depended strongly on the orientation of the fibre axis relative to the magnetic field, indicating that the essential light chain (ELC) and the neck were ordered. The probes were undergoing rapid motion within a cone. The half-width of the cone was estimated to be 65+/-5 degrees (SD, n=8). Addition of ADP affected little the hyperfine splitting and the angular spread of the probe distribution. In the presence of ADP and orthovanadate the intensity of the spectra decreased, which showed the dissociation of S1 and this was accompanied with the disappearance of the orientation dependence.     

Crystal Structure of Monomeric Photosystem II from Thermosynechococcus elongatus at 3.6-? Resolution

The membrane-embedded photosystem II core complex (PSIIcc) uses light energy to oxidize water in photosynthesis. Information about the spatial structure of PSIIcc obtained from x-ray crystallography was so far derived from homodimeric PSIIcc of thermophilic cyanobacteria. Here, we report the first crystallization and structural analysis of the monomeric form of PSIIcc with high oxygen evolution capacity, isolated from Thermosynechococcus elongatus. The crystals belong to the space group C222₁, contain one monomer per asymmetric unit, and diffract to a resolution of 3.6 Å. The x-ray diffraction pattern of the PSIIcc-monomer crystals exhibit less anisotropy (dependence of resolution on crystal orientation) compared with crystals of dimeric PSIIcc, and the packing of the molecules within the unit cell is different. In the monomer, 19 protein subunits, 35 chlorophylls, two pheophytins, the non-heme iron, the primary plastoquinone Q_A, two heme groups, 11 β-carotenes, 22 lipids, seven detergent molecules, and the Mn₄Ca cluster of the water oxidizing complex could be assigned analogous to the dimer. Based on the new structural information, the roles of lipids and protein subunits in dimer formation of PSIIcc are discussed. Due to the lack of non-crystallographic symmetry and the orientation of the membrane normal of PSIIcc perpendicular (∼87°) to the crystallographic b-axis, further information about the structure of the Mn₄Ca cluster is expected to become available from orientation-dependent spectroscopy on this new crystal form.     

Hyperfine structure of the photoexcited triplet state 3P680 in plant PS II reaction centres as determined by pulse ENDOR spectroscopy

The triplet states in plant photosystem II (PS II), 3P680, and from chlorophyll a, 3Chl a, in organic solution have been investigated using pulse ENDOR combined with repetitive laser excitation at cryogenic temperature with the aim to obtain their hyperfine (hf) structure. The large zero field splitting (ZFS) tensor of 3P680 enabled orientation selection via the electron spin resonance (EPR) field setting along the ZFS tensor axes. ENDOR spectra have been obtained for the first time also for the in-plane X- and Y-orientations of the ZFS tensor. This allowed a full determination of the hf-tensors of the three methine protons and one methyl group of 3P680. Based on the orientations of the axes of these hf-tensors, a unique orientation of the axes of the ZFS tensor of 3P680 in the Chl a molecular frame was obtained. These data serve as a structural basis for determining the orientation of 3P680 in the PS II protein complex by EPR on single crystals (see M. Kammel et al. in this issue). The data obtained represent the first complete set of the larger hf-tensors of the triplet state 3P680. They reflect the spin density distribution both in the highest occupied (HOMO) and lowest unoccupied (LUMO) orbitals. The data clearly confirm that 3P680 is a monomeric Chl a species at low temperature (T=10 K) used, as has been proposed earlier based on D- and E-values obtained from EPR and optically detected magnetic resonance (ODMR) studies. Comparison with the hf data for the cation and anion radicals of Chl a indicates a redistribution of spin densities in particular for the LUMO orbital of the triplet states. The electron spin distribution in the LUMO orbital is of special interest since it harbours the excited electron in the excited P680 singlet state, from which light-induced electron transfer proceeds. Observed shifts of hf couplings from individual nuclei of 3P680 as compared with 3Chl a in organic solution are of special interest, since they indicate specific protein interactions, e.g. hydrogen bonding, which might be used in future studies for assigning 3P680 to a particular chlorophyll molecule in PS II.     

Role of the electrostatic interactions in pre-orientation of subunits in the formation of protein-protein complexes

Theoretical estimation of contribution of the electrostatic interactions to pre-orientation of ribonuclease subunits in process of complex formation was carried out. The subunit was considered as a multipole consisting of partial charges of all atoms of the molecule. The object of investigation was a system of two subunits with their centers of gravity fixed at some distance in vacuum. It was proposed that each subunit independently could rotate freely around its fixed center of gravity. The relative orientation states of the subunits in such system were searched at which the system has electrostatic energy minima (equilibrium states). In first approximation the equilibrium states were found using especially designed approximate method for electrostatic interaction energy calculation, which permitted to calculate and compare the energies of the system in 24(5) (approximately 8 10(6)) states with different mutual orientation of subunits. The angular coordinates of the found equilibrium states were further specified by calculation with gradient sliding method. Angular coordinates of the equilibrium states and the shapes of energy surface cuts along each coordinate angle were calculated also for the intersubunits distances diminished down to 50 angstroms. The dispersions of the angular coordinates of equilibrium states caused by heat movement (at T=300 degrees) and their changes with shortening the distance between centers of gravity of subunits were estimated. Mutual orientation of subunits in the equilibrium states of the system under consideration was found to be similar to their mutual orientations in complex. Also it was found that relaxation time of the system, caused by electrostatic interaction of subunits, after removing the system from an equilibrium state, is much less in vacuum than the mean time between their Brownian collisions at room temperature. It follows from these results that in the case of ribonuclease in vacuum the electrostatic interactions of its subunits must be strong enough to realize the effective pre-orientation of subunits during their Brownian approach from distances of the order 100 angstroms. Preliminary consideration taking into account the effect of surrounding water molecules on the electrostatic interactions of ribonuclease subunits showed that weakening of the interaction must be much less than in the case when one uses in its calculation the macroscopic dielectric permeability value equal to 80. So the results obtained for vacuum seem to be true for water solution also. More strict theoretical analysis of this problem will be carried out in the following publication.     

Observation of two orientations from rigor cross-bridges in glycerinated muscle fibers

The fluorescence polarization from rhodamine labels specifically attached to the fast-reacting thiol of the myosin cross-bridge in glycerinated muscle fibers has been measured to determine the angular distribution of the cross-bridges in different physiological states of the fibers as a function of temperature. To investigate the fibers at temperatures below 0 degree C, we have added glycerol to the bathing solution as an anti-freezing agent. We find that the fluorescence polarization from the rhodamine probe detects distinct angular distributions of the cross-bridges in isometric-active, rigor, MgADP, and low ionic strength relaxed fibers at 4 degrees C. We also find that the rigor cross-bridges in the presence of glycerol can maintain at least two distinct orientations relative to the actin filament, one dominant at temperatures T greater than 2 degrees C and another dominant at T less than -10 degrees C. MgADP cross-bridges in the presence of glycerol maintain approximately the same orientation for all temperatures investigated. The rigor cross-bridge orientation at T less than -10 degrees C is similar to both the MgADP cross-bridge orientation in the presence of glycerol and the active muscle cross-bridge orientation at 4 degrees C. These findings show that the rigor cross-bridge in the presence of glycerol has at least two distinct orientations while attached to actin: one of them dominant at high temperature, the other dominant at low temperature or when MgADP is present. The latter orientation resembles that present in isometric-active fibers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fast ignition of an inertial fusion target with a solid noncryogenic fuel by an ion beam

The burning efficiency of a preliminarily compressed inertial confinement fusion (ICF) target with a solid noncryogenic fuel (deuterium-tritium beryllium hydride) upon fast central ignition by a fast ion beam is studied. The main aim of the study was to determine the extent to which the spatial temperature distribution formed under the heating of an ICF target by ion beams with different particle energy spectra affects the thermonuclear gain. The study is based on a complex numerical modeling including computer simulations of (i) the heating of a compressed target with a spatially nonuniform density and temperature distributions by a fast ion beam and (ii) the burning of the target with the initial spatial density distribution formed at the instant of maximum compression of the target and the initial spatial temperature distribution formed as a result of heating of the compressed target by the ion beam. The threshold energy of the igniting ion beam and the dependence of the thermonuclear gain on the energy deposited in the target are determined.     

Simultaneous determination of intramolecular distance distributions and conformational dynamics by global analysis of energy transfer measurements.

Fluorescence energy transfer is widely used for determination of intramolecular distances in macromolecules. The time dependence of the rate of energy transfer is a function of the donor/acceptor distance distribution and fluctuations between the various conformations which may occur during the lifetime of the excited state. Previous attempts to recover both distance distributions and segmental diffusion from time-resolved experiments have been unsuccessful due to the extreme correlation between fitting parameters. A method has been developed, based on global analysis of both donor and acceptor fluorescence decay curves, which overcomes this extreme cross-correlation and allows the parameters of the equilibrium distance distributions and intramolecular diffusion constants to be recovered with high statistical significance and accuracy. Simulation studies of typical intramolecular energy transfer experiments reveal that both static and dynamic conformational distribution information can thus be obtained at a single temperature and viscosity.     

Fundamental parameters of a relativistic runaway electron avalanche in air

A relativistic runaway electron avalanche in air is simulated numerically by the Monte Carlo method with allowance for a large number of elementary processes involving electrons, positrons, and photons. The characteristic time scale of the avalanche amplification is calculated as a function of the overvoltage δ relative to the minimum value of the drag force between the electrons and the atomic particles of the medium. The dynamics of the formation of the electron energy distribution is investigated. The steady-state mean electron energy depends weakly on δ. Over a wide range of δ values, there exists a universal electron energy distribution, which is essentially independent of δ. The angular distributions of electrons integrated over energies, as well as the angular distributions for different energy groups, are calculated. Analytic approximations for the energy and angular distributions are obtained.     

Maximum-entropy calculation of free energy distributions for two forms of myoglobin

The temperature dependence of the heat capacity of myoglobin depends dramatically on pH. At low pH (near 4.5), there are two weak maxima in the heat capacity at low and intermediate temperatures, respectively, whereas at high pH (near 10.7), there is one strong maximum at high temperature. Using literature data for the low-pH form (Hallerbach and Hinz, 1999) and for the high-pH form (Makhatadze and Privalov, 1995), we applied a recently developed technique (Poland, 2001d) to calculate the free energy distributions for the two forms of the protein. In this method, the temperature dependence of the heat capacity is used to calculate moments of the protein enthalpy distribution function, which in turn, using the maximum-entropy method, are used to construct the actual distribution function. The enthalpy distribution function for a protein gives the fraction of protein molecules in solution having a given value of the enthalpy, which can be interpreted as the probability that a molecule picked at random has a given enthalpy value. Given the enthalpy distribution functions at several temperatures, one can then construct a master free energy function from which the probability distributions at all temperatures can be calculated. For the high-pH form of myoglobin, the enthalpy distribution function that is obtained exhibits bimodal behavior at the temperature corresponding to the maximum in the heat capacity (Poland, 2001a), reflecting the presence of two populations of molecules (native and unfolded). For this form of myoglobin, the temperature evolution of the relative probabilities of the two populations can be obtained in detail from the master free energy function. In contrast, the enthalpy distribution function for the low-pH form of myoglobin does not show any special structure at any temperature. In this form of myoglobin the enthalpy distribution function simply exhibits a single maximum at all temperatures, with the position of the maximum increasing to higher enthalpy values as the temperature is increased, indicating that in this case there is a continuous evolution of species rather than a shift between two distinct population of molecules.     

Application of optical diffractometry in studies of cell fine structure. Comparison of arterial smooth muscle cells in contractile and synthetic state

Arterial smooth muscle cells in contractile and synthetic state were analyzed by optical diffractometry. Cell sections (80-90 nm) were photographed in an electron microscope and diffraction patterns of the plates (negatives) were produced using a helium-neon laser. Radial and angular distributions of light intensity in the diffractograms were measured and digitized using an electronic detector plate consisting of ring- and wedge-shaped photosensitive elements; radial distributions provide information about size of structures and distances between them and angular distributions about spatial orientation of structures in the images. Micrographs of nuclei and cytoplasm were analyzed separately (40-50 plates in each group). Computerized statistical analysis of radial distributions of light intensity showed that the nuclear chromatin pattern differed between cells in contractile and synthetic state. The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 96.5% of the cases. Analysis of radial distributions of light intensity similarly showed marked differences in cytoplasmic structure between cells in contractile state (dominated by bundles of myofilaments) and synthetic state (dominated by cisternae of rough endoplasmic reticulum). The probability that the observed difference could have arisen purely by chance was less than 10(-5). Computer-aided classification to the a priori known cell group was correct in 92.0% of the cases. In contrast, analysis of angular distributions of light intensity did not indicate any statistically significant differences between contractile and synthetic state cells. A likely reason is that both myofilaments and cisternae of rough endoplasmic reticulum were arranged in parallel. The results demonstrate that optical diffractometry is a useful method for image analysis in studies of cell fine structure. It provides information about size and orientation of structures with poorly defined shape and is particularly well suited for studies on cell differentiation and effects of pharmacological and other experimental treatments on cell fine structure. It represents an alternative and a complement to stereology for quantitative and objective evaluation of morphological data.     

Orientation of spin-labeled light chain 2 of myosin heads in muscle fibers

Electron paramagnetic resonance (e.p.r.) spectroscopy has been used to monitor the orientation of spin labels attached rigidly to a reactive SH residue on the light chain 2 (LC2) of myosin heads in muscle fibers. e.p.r. spectra from spin-labeled myosin subfragment-1 (S1), allowed to diffuse into unlabeled rigor (ATP-free) fibers, were roughly approximated by a narrow angular distribution of spin labels centered at 66 degrees relative to the fiber axis, indicating a uniform orientation of S1 bound to actin. On the other hand, spectra from spin-labeled heavy meromyosin (HMM) were roughly approximated by two narrow angular distributions centered at 42 degrees and 66 degrees, suggesting that the LC2 domains of the two HMM heads have different orientations. In contrast to S1 or HMM, the spectra from rigor fibers, in which LC2 of endogenous myosin heads was labeled, showed a random orientation which may be due to distortion imposed by the structure of the filament lattice and the mismatch of the helical periodicities of the thick and thin filaments. However, spectra from the fibers in the presence of ATP analog 5'-adenylyl imidodiphosphate (AMPPNP) were approximated by two narrow angular distributions similar to those obtained with HMM. Thus, AMPPNP may cause the LC2 domain to be less flexible and/or the S2 portion to be more flexible, so as to release the distortion of the LC2 domain and make it return to its natural position. At high ionic strength, AMPPNP disoriented the spin labels as ATP did under relaxing conditions, suggesting that the myosin head is detached from and/or weakly (flexibly) attached to a thin filament.     

Induced CD of DNA intercalators: electric dipole allowed transitions

The induced CD of an electric dipole allowed transition of a DNA intercalator has been calculated using the “matrix method” and a set of DNA ππ* transitions recently adopted for calculating the CD of DNA by Rizzo and Schellman [(1984) Biopolymers 23 , 435–470]. The induced CD is strongly dependent on the angular orientation of the intercalator and only moderately dependent on its location within the intercalation pocket. The dependence of the CD on the orientation and location of the intercalator was studied for some representative conformations of di- and tetranucleotide duplexes of (dGdC) and (dAdT). The effect of alternative DNA transition moment directions was also tested. The orientation dependence compares well with the previously predicted 1-2 cos² γ dependence [B. Nordén and F. Tjerneld (1982) Biopolymers 21 , 1713–1734]. Measured induced CD spectra of methylene blue (MB) intercalated in double-stranded poly(dAdT), poly(dGdC), and calf-thymus DNA are discussed on the basis of the results of the calculations. Major differences between the induced CD spectra are likely to reflect different modes of intercalation for the different nucleotide sequences. In particular, the concluded geometry in solution for MB intercalated in poly(dAdT) differs significantly from the corresponding geometry found in dinucleotide–intercalator crystals.     

SAR versus Sinc: What is the appropriate RF exposure metric in the range 1–10 GHz? Part I: Using planar body models

This is the first of two articles addressing the most appropriate crossover frequency at which incident power flux density (S_inc) replaces the spatial peak value of the specific energy absorption rate (SAR) averaged over 1 or 10 g (i.e., peak 1 or 10 g SAR) as the basic restriction for protecting against radiofrequency (RF) heating effects in the 1–10 GHz range. Our general approach has been to compare the degree of correlation between these basic restrictions and the peak induced tissue temperature rise (ΔT) for a representative range of population/exposure scenarios. In this article we particularly address the effect of human population diversity in the thickness of body tissue layers at eight different sites of the body. We used a Monte Carlo approach to specify 32000 models (400 models for each of 8 body sites for 10 frequencies) which were representative of tissue thicknesses for age (18–74 years) and sex at the eight body sites. Histogram distributions of S_inc and peak 1 and 10 g SAR corresponding to a peak 1 °C temperature rise were obtained from RF and thermal analyses of 1D multiplanar models exposed to a normally incident plane wave ranging from 1 to 10 GHz in thermo‐neutral environmental conditions. Examination of the distribution spread of the histograms indicated that peak SAR was a better predictor of peak tissue temperature rise across the entire 1–10 GHz frequency range than S_inc, as indicated by the smaller spread in its histogram distributions, and that peak 10 g SAR was a slightly better predictor than peak 1 g SAR. However, this result must be weighed against partly conflicting indications from complex body modeling in the second article of this series, which incorporates near‐field effects and the influence of complex body geometries. Bioelectromagnetics 31:454–466, 2010. © 2010 Wiley‐Liss, Inc.     

Kinetics of the interaction of dihydroalprenolol with beta-adrenergic receptors in rat cerebral cortex

Time and temperature dependence of the binding of 3H-dihydroalprenolol (3H-DHA) to beta-adrenergic receptors in rat cerebral cortex is described. The kinetic data obtained suggest that 3H-DHA binding proceeds through a two-step reaction scheme consisting of a bimolecular association step followed by an unimolecular internal conversion of the radioligand receptor complex (isomerisation). Equilibrium thermodynamic analysis provided evidence that the over-all binding process is associated with a small decrease in enthalpy and a substantial increase in entropy. Within the framework of the two-step binding kinetics, the evaluation of the temperature dependence by the van't Hoff analysis resulted in values for thermodynamic parameters for the single equilibrium steps. The data suggest that the association step can be considered as a bimolecular hydrophobic interaction which is mainly entropy-driven due to the release of structural water, while the isomerisation step is accompanied by a large negative change in both enthalpy and entropy. The large negative change in the activation entropy for the forward reaction of the isomerisation step, obtained from evaluation of Arrhenius plots, indicates an internal conversion to a highly ordered receptor-ligand complex, while the low activation energy points to a small threshold energy for reaching this structure. Thus, these result support a previous assumption that the hydrophobic center of an adrenergic antagonist interacts with the receptor by entering a pocket (Cherksey et al. 1981).     

DNA complex lesions induced by protons and ⋅-particles: track structure characteristics determining linear energy transfer and particle type dependence

The yield of DNA double-strand breaks (dsb) and DNA complex lesions induced by protons and α-particles of various energies was simulated using a Monte Carlo track structure code (MOCA15) and a simple model of the DNA molecule. DNA breaks of different complexity were analysed. The linear energy transfer (LET) and particle-type dependence of lesions of higher complexity seems to confirm the importance of clustered damage in DNA as a relevant step leading to biological endpoints such as cell inactivation. The detailed structure of proton and α-particle tracks was analysed to identify the main characteristics possibly responsible for such a dependence. The role of the primary ion and of its secondary electrons in inducing dsb and complex lesions is described, showing that the relative contribution of secondary electron tracks alone in inducing clustered lesions is almost negligible at high LET, but tends to dominate below ≈10 keV/μm. This is consistent with the observed similar effectiveness of low-LET fast particle radiation and sparsely ionizing radiation such as x-rays. The dependence on LET and particle type is mainly due to energy deposition events of the primary ion together with short range electrons surrounding the ion track; the yield of complex lesions due to secondary electron tracks alone is substantially LET independent. The radial distributions of the energy contributing to the induction of complex lesions were analyzed and compared with the radial distributions of energy deposition of the full tracks. The results suggest that the stochastic behaviour (i.e. cluster properties) of the energy deposition pattern within a radius of a few nanometers around the ion track plays a relevant role in determining the biological radiation effectiveness. Received: 20 December 1996 / Accepted in revised form: 5 March 1997     

Orientational distribution of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles as determined by global analysis of frequency domain fluorimetry data

Polarized differential phase and modulation ratios were obtained for 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles by using multifrequency phase fluorometry. Data were analyzed in terms of both empirical sums-of-exponentials modeling and directly in terms of the orientational distribution functions. The orientational analysis model was used to recover the angular distribution of DPH and the rotational diffusion coefficient in the various membrane model systems throughout the phase transition. A global analysis methodology was utilized to obtain an internally consistent set of parameters that fit all of the data simultaneously. The rank order parameters (P2) and (P4) were extracted from the experimental data, and the angular distribution functions of DPH were calculated. When the time-zero anisotropy (r0) of several sets of data taken at various temperatures were linked in a single global analysis, better recovery of the fourth rank order parameter (P4), diffusion constant D, and r0 was obtained with respect to the unlinked analysis. From these recovered values, a detailed picture concerning the orientational distribution of DPH in membranes as a function of temperature was obtained. The results suggest that a single population of DPH molecules was present in the bilayers with their orientational distributions dependent upon the physical state of the membranes in the pure phases. During the phase transition, a superposition of two populations corresponding to the population of the pure phases was present. As the temperature increased in the transition region, one population was increasing at the expense of the other.     

Energetics of ribonuclease A catalysis. 3. Temperature dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Studies of the temperature dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate at pH 5.0 are reported. Contributions to the temperature dependence of the apparent Michaelis-Menten parameters from temperature-sensitive protonic equilibria (primarily the coupled protonation/deprotonation of the active-site histidine residues) were included in our analysis. The data were interpreted by employing a transition-state approach. By comparing the temperature dependence of the rate constant for the nonenzymatic hydrolysis of the substrate with the temperature dependence of the enzyme-catalyzed reaction, we obtained values for the enthalpy change, entropy change, and heat capacity change for the interaction of the reaction transition state with the enzyme. These thermodynamic quantities were then interpreted by comparison with corresponding values for the binding of cytidine 2'- and 3'-phosphate to the enzyme. A model is presented for the enzyme-transition-state interaction involving the favorable transfer of a proton from the transition state to a histidine residue at the active site and the formation of hydrogen bonds and van der Waals contacts between the pyrimidine ring of the transition state and the enzyme's binding pocket. These elementary interactions are consistent with the determined values of the enthalpy change and entropy change, as well as earlier reported ionic strength and solvent isotope dependence studies. The Gibbs energy contributions from these elementary interactions have also been estimated, giving a sum approximately equal to the experimentally determined value for the stabilization energy of the enzyme-transition-state complex. The model thus provides an explanation for the magnitude of the approximately 10(10)-fold rate enhancement achieved by this enzyme.